Methods of stimulating an immune response against prostate specific antigen

ABSTRACT

The invention provides a prostate specific antigen oligo-epitope peptide (PSA-OP) that is useful as an immunogen in the prevention or treatment of prostatic cancer and in the inhibition of prostatic cancer cells and in the establishment and characterization of PSA-specific cytotoxic T-cell lines. In particular, the invention provides methods for eliciting an immune response against PSA comprising administering (i) a priming inoculation of a first recombinant virus encoding PSA-OP and (ii) one or more boosting inoculations of a second recombinant virus encoding PSA-OP, wherein the first and second recombinant viruses are from a different genus.

FIELD OF THE INVENTION

The present invention relates generally to generation of cellular andhumoral immune responses to a mammalian prostate-specific antigen (PSA).More specifically, the present invention relates to a prostate specificantigen (PSA) oligo-epitope peptide useful in generating PSA specific Tlymphocytes for prevention or treatment of prostate cancer.

BACKGROUND OF THE INVENTION

Cancer of the prostate is the most commonly diagnosed cancer in men andis the second most common cause of cancer death (Carter et al, 1990;Armbruster et al, 1993). If detected at an early stage, prostate canceris potentially curable. However, a majority of cases are diagnosed atlater stages when metastasis of the primary tumor has already occurred(Wang et al, 1982). Even early diagnosis is problematic because not allindividuals who test positive in these screens develop cancer. Presenttreatment for prostate cancer includes radical prostatectomy, radiationtherapy, or hormonal therapy. No systemic therapy has clearly improvedsurvival in cases of hormone refractory disease. With surgicalintervention, complete eradication of the tumor is not always achievedand the observed re-occurrence of the cancer (12-68%) is dependent uponthe initial clinical tumor stage (Zietman et al, 1993). Thus,alternative methods of treatment including prophylaxis or prevention aredesirable.

Prostate specific antigen (PSA) is a 240 amino acid member of theglandular kallikrein gene family. (Wang et al, 1982; Wang et al, 1979;Bilhartz et al, 1991). PSA is a serine protease, produced by normalprostatic tissue, and secreted exclusively by the epithelial cellslining prostatic acini and ducts (Wang et al, 1982; Wang et al, 1979;Lilja et al, 1993). Prostatic specific antigen can be detected at lowlevels in the sera of healthy males without clinical evidence ofprostate cancer. However, during neoplastic states, circulating levelsof this antigen increase dramatically, correlating with the clinicalstage of the disease (Schellhammer et al, 1993; Huang et al, 1993; Kleeret al, 1993; Oesterling et al, 1991). Prostatic specific antigen is nowthe most widely used marker for prostate cancer. The tissue specificityof this antigen makes PSA a potential target antigen for active specificimmunotherapy (Armbruster et al, 1993; Brawer et al, 1989), especiallyin patients who have undergone a radical prostatectomy in which the onlyPSA expressing tissue in the body should be in metastatic deposits.Recent studies using in-vitro immunization have shown the generation ofCD4 and CD8 cells specific for PSA (Peace et al, 1994; Correale et al,1995). However, although weak natural killer cell responses have beenoccasionally documented in prostate cancer patients (Choe et al, 1987),attempts to generate an in vivo immune response have met with limitedsuccess. For example, several attempts to actively immunize patientswith prostate adenocarcinoma cells admixed with Bacillus Calmette-Gurein(BCG) have shown little or no therapeutic benefit (Donovan et al, 1990).The ability to elicit an immune response as a result of exposure to PSAin vivo would be extremely useful.

Vaccinia virus has been used in the world-wide eradication of smallpox.This virus has been shown to express a wide range of inserted genes,including several tumor associated genes such as p97, HER2/neu, p53 andETA (Paoletti et al, 1993). Other pox viruses that have been suggestedas useful for expression of multiple genes include avipox such as fowlpox. Cytokines expressed by recombinant vaccinia virus include IL-1,IL-2, IL-5, IL-6, TNF-α and IFN-γ (Paoletti et al, 1993). Recombinantpox viruses, for example vaccinia viruses, are being considered for usein therapy of cancer because it has been shown in animal models that theco-presentation of a weak immunogen with the highly immunogenic poxvirusproteins can elicit a strong immune response against the inserted geneproduct (Kaufman et al, 1991, Paoletti et al, 1993; Kantor et al, 1992a;Kantor et al, 1992b; Irvine et al, 1993; Moss et al, 1993). Arecombinant vaccinia virus containing the human carcinoembryonic antigengene has just completed phase 1 clinical trials in carcinoma patientswith no evidence of toxicity other than that observed with the wild typesmallpox vaccine (Kantor et al, 1992b).

Currently, models for the evaluation of prostate therapeutics includethe canine (McEntee et al, 1987) and the Dunning rat (Isaacs et al,1986); neither of these models, however, are practical for the study ofPSA-recombinant vaccines due to the very low homology of rat and caninePSA to human PSA (Karr et al, 1995; Schroder et al, 1982). In contrast,the prostate gland of the rhesus monkey is structurally and functionallysimilar to the human prostate (Wakui et al, 1992). At the molecularlevel there is 94% homology between either the amino acid or nucleicacid sequences of rhesus PSA (Gauther et al, 1993) and those sequencesof human prostate specific antigen (Karr et al, 1995; Lundwall et al,1987). Thus, human PSA is essentially an autoantigen in the rhesusmonkey. Accordingly, the rhesus monkey can serve as a model forautologous anti-PSA immune reactions.

Since PSA shares extensive homology with members of the kallikrein genefamily which are expressed in normal tissue, it is important to useminimal epitope peptides to avoid unwarranted cross reactivity. Theseepitopes have been selected for their divergence with members of thekallikrein gene family.

Studies disclosed in U.S. Ser. No. 08/500,306 have shown that two PSAepitope peptides (PSA-1 and PSA-3), 10-mers selected to conform to humanHLA class 1-A2 motifs, can elicit CTL responses in both normal donorsand patients with prostate cancer. (Correale et al, 1995) The presentinvention discloses the advantage of PSA-oligo-epitope peptidescomprising more than one PSA epitope peptide in generating PSA specificcellular immune responses.

SUMMARY OF THE INVENTION

The invention is a prostate specific antigen oligo-epitope peptide andanalogs thereof which are immunogenic amongst individuals with at leastone HLA-class I allele, preferably more than one HLA-class I allele. Theprostate specific antigen oligo-epitope peptide comprises more than one8 to 12 mer PSA epitope peptide adjoined together, each 8 to 12 mer PSAepitope peptide binds to a human HLA class I molecule type. The 8 to 12mer PSA epitope peptides may be adjoined via a short amino acidsequence.

The prostate specific antigen oligo-epitope peptide and analogs thereofelicit PSA specific cytotoxic T lymphocytes which lyse cells havingbound thereto PSA, fragments of PSA, or one or more PSA epitope peptidesthereof.

Another object of the invention is a pharmaceutical compositioncomprising a prostate specific antigen oligo-epitope peptide or analogsthereof and a pharmaceutically acceptable carrier. The pharmaceuticalcomposition is useful as an immunogen and as a therapeutic in theprevention or treatment of prostate cancer and in inhibiting growth ofPSA⁺ cancer cells.

Another aspect of the invention is a method of generating PSA specificcytotoxic T lymphocytes by in vivo administration of an effective amountof a prostate specific antigen oligo-epitope peptide or analogs thereof,alone or in combination with an adjuvant or liposomes. The PSA specificcytotoxic T lymphocytes which arise from immunization are useful inmethods of inhibiting or killing PSA positive tumor cells in a mammal.

Yet another aspect of the invention is a method of generating PSAspecific cytotoxic T lymphocytes in vitro by stimulation of lymphocytesfrom a source with an effective amount of a prostate specific antigenoligo-epitope peptide or analogs thereof, alone or in combination withone or more cytokines to generate PSA specific cytotoxic T lymphocytes.Such PSA specific cytotoxic T lymphocytes may be adoptively transferredinto a mammal for the prevention or treatment of prostate cancer and forinhibiting or killing PSA positive tumor cells.

A further object of the invention is a DNA sequence encoding a prostatespecific antigen oligo-epitope peptide comprising more than one 8 to 12mer PSA epitope peptide or analogs thereof, each 8 to 12 mer PSA epitopepeptide binds to a human HLA class I molecule type.

An object of the invention is a vector comprising at least one insertionsite containing a DNA sequence encoding a prostate specific antigenoligo-epitope peptide or analogs thereof, operably linked to a promotercapable of expression in a host cell.

Another aspect of the invention is a method of generating PSA specificcytotoxic T lymphocytes by administration into a mammalian host aneffective amount of a recombinant virus vector comprising at least oneinsertion site containing a DNA sequence encoding a prostate specificantigen oligo-epitope peptide or analogs thereof.

We have discovered that by using a recombinant viral vector, preferablya pox virus vector having at least one insertion site containing a DNAsegment encoding prostate-specific antigen (PSA), or a cytotoxic T-celleliciting epitope thereof, operably linked to a promoter capable ofexpression in the host, a specific humor and cellular immune response toPSA can be generated. The method preferably comprises introducing asufficient amount of the recombinant pox virus vector into a host tostimulate the immune response, and contacting the host with additionalPSA at periodic intervals thereafter. The additional PSA, or a cytotoxicT-cell eliciting epitope thereof, may be added by using a second poxvirus vector from a different pox genus. In another embodiment,additional PSA can be added by contacting the host with PSA by a varietyof other methods, including in one preferred embodiment adding PSA. ThePSA may be formulated with an adjuvant or in a liposomal formulation.

In a further embodiment, an immune response to PSA can be generated bycontacting the host initially with a sufficient amount of PSA, or acytotoxic T-cell eliciting epitope thereof, to stimulate an immuneresponse and at periodic intervals thereafter contacting the host withadditional PSA. The additional PSA, or a cytotoxic T-cell generatingfragment thereof, may be added using a pox virus vector as discussedabove.

We have also discovered that human cytotoxic T-cells specific for PSAcan be produced using a cytotoxic T-cell eliciting epitope of the PSAand that these cells have the ability to lyse PSA-expressing humanprostate carcinoma cells.

As used herein the term “prostate specific antigen” includes the nativeprotein whether purified from a native source or made by recombinanttechnology, as well as any polypeptide, mutein, or portion derivedtherefrom that is capable of generating an immune response to a nativeconformationally correct PSA. For example, one can make conservativeamino acid substitutions in the molecule without adversely affecting theability to use the recombinant to generate an antibody that will alsorecognize native PSA.

The pox virus is preferably selected from the group of pox virusesconsisting of suipox, avipox, capripox and orthopox virus. Preferredorthopox include vaccinia, rabbit pox and raccoon pox. Preferred avipoxincludes fowlpox, canary pox and pigeon pox. A more preferred avipox isfowlpox. The preferred suipox is swinepox.

Vaccinia viral vectors may elicit a strong antibody response. Thus,while numerous boosts with vaccinia vectors are possible, its repeateduse may not be preferred in certain instances. We have discovered thatby using pox from different genera to boost, this sensitivity problemcan be minimized. In accordance with the present invention, in order toavoid such problems, preferably, when the first or initial pox virusvector is vaccinia, the second and subsequent pox virus vectors areselected from the pox viruses from a different genus such as suipox,avipox, capripox or an orthopox immunogenically distinct from vaccinia.

Adjuvants include, for example, RIBI Detox, QS21, alum and incompleteFreund's adjuvant. Liposomal formulations can also be used.

Human cytotoxic T-cells specific for PSA produced in accordance with thepresent invention can be isolated from a human host. These cells can beused in drug assays, used to map cytotoxic T-cells eliciting antigenepitopes or in adoptive cell therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Western blot of PSA from rV-PSA infected BSC-40 cells.Lanes 2-4 are extracts from supernatant fluid from cells infectedovernight with rV-PSA at an MOI of 1, while Lanes 7-9 are extracts fromthe corresponding infected cells. Lanes 1 and 6 are supernatant extractsand cell extracts from V-Wyeth infected cells. Blot was developed usinga specific MAb for human PSA. This blot illustrates that cells infectedwith rV-PSA authentically express and secrete the 33 kD PSA protein.

FIGS. 2A, 2B and 2C show the manifestation of rV-PSA immunization. InFIG. 2A, the area of lesions was measured 7 days following eachinoculation of rhesus monkeys with either V-Wyeth (open circles) orrV-PSA (closed circles). In FIG. 2B, the duration of the lesion wasmonitored as time of scab disappearance. In FIG. 2C, the extent of lymphnode swelling was recorded and characterized as very swollen (3+), i.e.more than two axillary nodes swollen; swollen (2+), i.e. one or twonodes easily palpable; marginally swollen (1+), i.e. one node was barelypalpable or not swollen (O), 7 days following inoculation with vacciniavirus. Each symbol represents one monkey.

FIG. 3 shows the amino acid sequence of a prostate specific antigenoligo-epitope peptide.

FIG. 4 shows the nucleic acid sequence encoding a prostate specificantigen oligo-epitope peptide.

FIG. 5 shows the nucleic acid sequence of an insert (within the Kpn-1site and the Xho-1 site) cloned into recombinant vaccinia comprising thenucleic acid sequence encoding a prostate specific antigen oligo-epitopepeptide (PSA AA490-594), an endoplasmic reticulum trafficking signal(E3/19K signal), and a universal T helper epitope peptide, tetanustoxoid CD4 epitope (TT CD4 epitope). The 5→3′ insert has SEQ. ID NO.:14. The complementary 3→5′ insert has SEQ. ID NO.: 15.

DETAILED DESCRIPTION OF THE INVENTION

The invention is a prostate specific antigen (PSA) oligo-epitopepeptide. The prostate specific antigen oligo-epitope peptide ischaracterized by its ability to elicit a cellular immune responsespecific against PSA or portion thereof and against cells expressing orbinding PSA or portion thereof in a mammalian host.

In general, tumor associated antigen proteins, such as PSA protein, areprocessed by intracellular proteases into small epitope peptides whichare then transported to the cell surface bound tightly in a cleft on theHLA class I molecule. T cells recognize these small epitope peptidesonly when they are bound within this cleft on the target cell. The classI molecules of the major histocompatibility complex (MHC) are found onthe surfaces of cells including tumor cells and are the self moleculesrecognized in conjunction with antigen by cytotoxic T cells.

As disclosed herein, short PSA epitope peptides composed of an aminoacid sequence of about 9 to 10 amino acids bind directly within thecleft of an HLA class I molecule without intracellular processing. Twosuch PSA epitope peptides, PSA-1 and PSA-3 are bound by one specific HLAclass I molecule type, i.e. HLA-class I-A2. Each of these individual PSAepitope peptides elicites PSA specific cytotoxic T cells which lyse PSA⁺HLA-class I-A2 target cells.

However, due to polymorphisms within the peptide binding cleft of classI molecules there are variations among individuals in the ability oftheir class I molecule types to bind antigens. Thus, while 9 mer and 10mer PSA epitope peptides are effective in generating cytotoxic Tlymphocytes in individuals having a HLA-class I A2 type, the same 9 merand 10 mer PSA epitope peptides may not be effective or may be lesseffective in generating cytotoxic T lymphocytes in individuals havingdifferent interactions of HLA-peptide complexes with different T-cellreceptor molecules, which can differ amongst individuals.

The prostate specific antigen oligo-epitope peptide and analogs thereofof the present invention which comprises more than one 8 to 12 mer PSAepitope peptides linked together has the advantage of eliciting cellularimmune responses in individuals having diverse HLA class I moleculetypes or alleles. The prostate specific antigen oligo-epitope peptide isprocessed by extracellular proteases or other mechanisms allowing theresulting PSA epitope peptide cleavage fragments to bind to the cleft ofthe same or several different HLA class I molecule types.

Thus, the prostate specific antigen oligo-epitope peptide, in contrastto shorter individual PSA epitope peptides of about 8 to 12 mer, isimmunogenic for a broad segment of the human population with differingHLA class I molecule types and differing T-cell receptor interactionswith HLA class-I peptide complexes.

The prostate specific antigen oligo-epitope peptide comprises peptidesequences which fit the consensus motif for at least one HLA class Imolecule type, and preferably contains peptide sequences that fit theconsensus motif of more than one HLA class I molecule type.

In one embodiment, the prostate specific antigen oligo-epitope peptidecomprises more than one 8 to 12 mer PSA epitope peptide sequence whichfits the consensus motif for one or more of the HLA class I moleculetypes, which include but are not limited to HLA-A1, HLA-A2, A3, All,HLA-A24, HLA-A26, HLA-A28, HLA-A32, HLA-B7, HLA-B44, HLA-Cw3, HLA-Cw4,HLA-Cw5, Aw68 and B53.

In another embodiment, the prostate specific antigen oligo-epitopepeptide comprises PSA peptide sequences which fit the consensus motiffor HLA-A2, A.3, A11 and B53. HLA class I molecule type, HLA-A3 ispresent in 26 and 17% of North American caucasians and blacksrespectively. HLA class I molecule type, HLA-A11 is present in 40% ofthe Asian population, and HLA-53 is present in 22% of blacks. Thus, theprostate specific antigen oligo-epitope peptide is useful in generatinga cellular immune response against PSA in a broad segment of the humanpopulation with differing HLA class I molecule types.

Individual 9 mer and 10 mer amino acid PSA epitope peptides are capableof generating cytotoxic T lymphocytes (CTLs) in vitro and capable ofpulsing target cells for lysis by PSA-specific CTLs. Modeling studieshave shown that the individual 9 mer or 10 mer PSA epitope peptides fitsinto the groove of a unique class I HLA molecule, A2. The presentinvention of linking various combinations of 8 to 12 mer PSA epitopepeptides allows for one immunogen instead of two or more separateimmunogens, as the oligo-epitope peptide is efficiently processed byproteases at the antigen presenting cell surface or target cell surface,or processed by other mechanisms to form appropriate smaller PSA epitopepeptide cleavage fragments that interact or bind with the same class Imolecule type or with a variety of class I molecule types resulting inthe generation of a PSA specific cellular response in the majority ofthe human population.

The PSA oligo-epitope peptide or analogs thereof generate cytotoxic Tlymphocytes which inhibit or lyse target cells which express or havebound thereto PSA and target cells which express or have bound theretoone, preferably more than one 8 to 12 mer PSA epitope peptides.Additionally, target cells inhibited or lysed by the cytotoxic Tlymphocytes may have one HLA class I molecule type or a plurality HLAclass I molecule types.

The prostate specific antigen oligo-epitope peptide, analog orfunctional equivalent thereof comprise more than one 8 to 12 mer PSAepitope peptide which conforms to at least one human HLA class Imolecule type, preferably more than one human HLA class I moleculetypes. A first PSA epitope peptide that conforms to a human HLA class Imolecule type is adjoined to a second PSA epitope peptide that conformsto a human HLA class I molecule type. The first and second epitopepeptides may be joined together directly or optionally joined togethervia a short amino acid linker sequence. The peptides are joined togetherby peptide bonds.

The individual PSA epitope peptides which comprise the prostate specificantigen oligo-epitope peptide may each vary in the number of amino acidsbut typically comprise about 8 to about 12 amino acids, preferably about9 to about 10 amino acids. In one embodiment, the first and second PSAepitope peptide each comprise about 10 amino acids.

The prostate specific antigen oligo-epitope peptide may compriserepeating units of the individual PSA epitope peptide, or combinationsof repeating units. The total number of repeating units in the prostatespecific antigen oligo-epitope peptide of individual PSA epitopepeptides or combinations of PSA epitope peptides may be about 6 to about8.

When utilized, the amino acid linker sequence for joining the 8 to 12mer PSA epitope peptides comprises about 1 to about 10 amino acids,preferably about 1 to about 5 amino acids. In one embodiment, the linkercomprises about three amino acids. In a particular embodiment, thelinker sequence comprises Asp-His-Leu. The amino acid linker sequence iscleavable by proteolytic activity.

In one embodiment, the prostate specific antigen oligo-epitope peptidecomprises one or more PSA1 peptides having SEQ. ID No.: 1 or analogsthereof and one or more PSA2 peptides having SEQ. ID No.: 2 or analogsthereof. In a preferred embodiment, the prostate specific antigenoligo-epitope peptide comprises one PSA1 peptide and one PSA2 peptidelinked together.

Additionally, the prostate specific antigen oligo-epitope peptidecomprises one or more additional PSA epitope peptides adjoined to thesecond PSA epitope peptide. In one embodiment, a third PSA peptidecomprises an amino acid sequence which overlaps the sequence at thecarboxyl terminus of the second PSA epitope peptide. The overlap insequence may be by one to three amino acids. In one embodiment, theoverlap is by two amino acids. In a particular embodiment, a third PSAepitope peptide comprises QVHPQKVTK (SEQ. ID NO.: 3) in which theoverlapping sequence is QV.

In a preferred embodiment, the oligo-epitope peptide comprises the aminoacid sequence:

FLTPKKLQCVDLHVISNDVCAQVHPQKVTK (Seq. ID No.: 4) and analogs and variantsthereof. In one embodiment, the prostate specific antigen oligo-epitopepeptide comprises analogs with substitutions that include but are notlimited to valine at one or more positions at amino acid residuepositions 148, 149, 160 and/or 161. In yet another embodiment, theprostate specific antigen comprises analogs with deletions that includebut are not limited to deletion of one or more amino acids at positions151, 152 and 153.

The prostate specific antigen oligo-epitope peptide may be obtained byrecombinant DNA technology, by chemical peptide synthesis or byappropriate protease cleavage of the isolated, natural PSA.

The prostate specific antigen oligo-epitope peptide or analogs thereofof the present invention may be formulated into a pharmaceuticalcomposition in combination with a pharmaceutically acceptable carrierfor use as an immunogen in a mammal, preferably a human or primate. Thecomposition may further comprise one or more other constituents toenhance the immune response which include but are not limited tobiological response modifiers such as interleukin 2, interleukin 6,interleukin 12, interferon, tumor necrosis factor, GM-CSF andcyclophosphamide.

The prostate specific antigen oligo-epitope peptide is administered to amammal in an amount effective in generating a PSA specific cellularimmune response. The efficacy of the prostate specific antigenoligo-epitope peptide as an immunogen may be determined by in vivo or invitro parameters as are known in the art. These parameters include butare not limited to antigen specific cytotoxicity assays, regression ofPSA⁺ tumors, inhibition of PSA⁺ cancer cells, production of cytokinesand the like.

The prostate specific antigen oligo-epitope peptide may be administeredin a dose of about 0.5 mg to about 100 mg per kilogram body weight ofthe mammal. Several doses may be provided over a period of weeks asindicated. The PSA-OP or analogs thereof may be administered alone or incombination with adjuvants, liposomes, cytokines, biological responsemodifiers, or other reagents in the art that are known to enhance immuneresponses.

The PSA-OP may also be conjugated to other helper peptides or to largercarrier molecules to enhance the immunogenicity of the peptide. Thesemolecules include but are not limited to influenza peptide, tetanustoxoid, tetanus toxoid CD4 epitope, Pseudomonas exotoxin A,poly-L-lysine, and the like.

The invention also provides a method of generating PSA specificcytotoxic T lymphocytes in vivo or in vitro by stimulation oflymphocytes from a source with an effective amount of a prostatespecific antigen oligo-epitope peptide alone or in combination with oneor more cytokines in context of an HLA class I molecule to generate PSAspecific cytotoxic T lymphocytes. The sources of lymphocytes include butare not limited to peripheral blood lymphocytes, tumor infiltratinglymphocytes, lymph nodes and the like.

The invention encompasses a DNA sequence and analog and variant thereofwhich encodes a prostate specific antigen oligo-epitope peptide oranalog thereof. In one embodiment the DNA sequence comprises:

5′-TTC TTG ACC CCA AAG AAA 3′-AAG AAC TGG GGT TTC TTT    Phe Leu Thr ProLys Lys    CTT CAG TGT GTG GAC CTC    GAA GTC ACA CAC CTG GAG    Leu GlnCys Val Asp Leu    CAT GTT ATT TCC AAT GAC    GTA CAA TAA AGG TTA CTG   His Val Ile Ser Asn Asp    GTG TCT GCG CAA GTT CAC    CAC ACA CGC GTTCAA GTG    Val Cys Ala Gln Val His    CCT CAG AAG GTG ACC AAG-3′ (SEQ.ID NO.: 5)    GGA GTC TTC CAC TGG TTC-5′ (SEQ. ID NO.: 6)    Pro Gln LysVal Thr Lys (SEQ. ID NO.: 4)and analogs and variants thereof.

Included in the ambit of the invention are substitutions and deletionswithin the DNA sequence provided that the modifications result in afunctionally equivalent PSA-OP peptide or a peptide with enhancedimmunogenicity. In one embodiment a DNA sequence is substituted with theappropriate nucleic acids that encode analogs of the prostate specificantigen oligo-epitope peptide based on codon degeneracy as are known inthe art. Other substitutions in the DNA sequence include but are notlimited to valine at one or more positions at amino acid residueposition 148, 149, 160 and/or 161.

The invention further encompasses vectors and plasmids comprising a DNAsequence, analog or variant thereof which encodes a prostate specificantigen oligo-epitope peptide. The vectors may further comprise one ormore DNA sequences encoding helper peptides or large carrier molecule(s)to enhance the immunogenicity of the PSA-OP. These additional DNAsequences encode molecules that include but are not limited to influenzapeptide, tetanus toxoid, tetanus toxoid CD4 epitope, Pseudomonasexotoxin A, poly-L-lysine, and the like. In one embodiment, the vectorfurther comprises a tetanus toxoid CD4 epitope.

Of particular interest are recombinant viral vectors comprising a DNAsequence, analog or variant thereof which encodes a prostate specificantigen oligo-epitope peptide or analogs thereof. In one embodiment, therecombinant viral vector further comprises a tetanus toxoid CD4 epitope.In a particular embodiment, the recombinant viral vector is recombinantvaccinia comprising a DNA sequence encoding a prostate specific antigenoligo-epitope peptide, a DNA sequence encoding endoplasmic reticulumtrafficking signal, and a DNA sequence encoding tetanus toxoid CD4epitope as depicted in FIG. 5.

Host cells which may express the DNA encoding the prostate specificantigen oligo-epitope peptide or analogs thereof carried by vectors orplasmids are prokaryotic and eukaryotic host cells and include but arenot limited to E. coli, Listeria, Bacillus species, COS cells, CV-1,Vero cells, BSC-40 cells, HuTk143 cells, chick embryo fibroblasts,prostatic cells, tumor cells, antigen presenting cells and the like.When the host cells is an antigen presenting cell or a target cell, thehost cell should additionally express a MHC class I molecule.

We have induced an immune response specific to PSA in the rhesus monkeymodel by placing the PSA gene into a recombinant viral vector, i.e. apox vector such as vaccinia virus.

Additionally, an immune response to PSA can be generated by contactingthe host initially with a sufficient amount of PSA, or a cytotoxicT-cell eliciting epitope thereof, to stimulate an immune response and atperiodic intervals thereafter contacting the host with additional PSA.The additional PSA, or a cytotoxic T-cell generating fragment thereof,may be added using a pox virus vector.

A DNA fragment encoding the open reading frame of human PSA can beobtained, for example, from total RNA extracted from the humanmetastatic prostate adenocarcinoma cell line, LNCaP.FGC (CRL 1740,American Type Cell Culture (ATCC), Rockville, Md.) by reversetranscriptase PCR using PSA specific oligonucleotide primers 5′TCTAGAAGCCCCAAGCTTACCACCTGCA 3′ (SEQ. ID. NO.: 16), 5′TCTAGATCAGGGGTTGGCCACGATGGTGTCCTTGATCCACT 3′ (SEQ. ID. NO.: 17). Thenucleotide sequence of the PSA cDNA has been published (Lundwall et al,1987).

Recombinant human PSA can be obtained using a baculovirus expressionsystem in accordance with the method of Bei et al, J. Clin. Lab. Anal.,9:261-268 (1995), the disclosure of which is herein incorporated byreference.

Viral Vector

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding the carcinoma self-associated antigenor cytotoxic T-cell eliciting epitope are known to the skilled artisanand involve, for example, homologous recombination between the viral DNAsequences flanking the DNA sequence in a donor plasmid and homologoussequences present in the parental virus (Mackett et al, Proc. Natl.Acad. Sci USA 79:7415-7419 (1982)). For example, recombinant viralvectors such as a pox viral vector can be used in delivering the gene.The vector can be constructed for example by steps known in the art,e.g. analogous to the methods for creating synthetic recombinants of thefowlpox virus described in U.S. Pat. No. 5,093,258, the disclosure ofwhich is incorporated herein by reference. Other techniques includeusing a unique restriction endonuclease site that is naturally presentor artificially inserted in the parental viral vector to insert theheterologous DNA.

Pox viruses useful in practicing the present invention include orthopox,suipox, avipox and capripox virus.

Orthopox include vaccinia, ectromelia and raccoon pox. The preferredorthopox is vaccinia.

Avipox includes fowlpox, canary pox and pigeon pox. The preferred avipoxis fowlpox.

Capripox include goatpox and sheeppox.

A preferred suipox is swinepox.

Other viral vectors that can be used include other DNA viruses such asherpes virus and adenoviruses, and RNA viruses such as retroviruses andpolio.

For example, the DNA gene sequence to be inserted into the virus can beplaced into a donor plasmid, e.g. an E. coli plasmid construct, intowhich DNA homologous to a section of DNA such as that of the insertionsite of the poxvirus where the DNA is to be inserted has been inserted.Separately the DNA gene sequence to be inserted is ligated to apromoter. The promoter-gene linkage is positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of pox DNA which isthe desired insertion region. With a parental pox viral vector, a poxpromoter is used. The resulting plasmid construct is then amplified bygrowth within E. coli bacteria and isolated. Preferably, the plasmidalso contains an origin of replication such as the E. coli origin ofreplication, and a marker such as an antibiotic resistance gene forselection and propagation in E. coli.

Second, the isolated plasmid containing the DNA gene sequence to beinserted is transfected into a cell culture, e.g. chick embryofibroblasts, along with the parental virus, e.g. poxvirus. Recombinationbetween homologous pox DNA in the plasmid and the viral genomerespectively results in a recombinant poxvirus modified by the presenceof the promoter-gene construct in its genome, at a site which does notaffect virus viability.

As noted above, the gene is inserted into a region (insertion region),in the virus which does not affect virus viability of the resultantrecombinant virus. The skilled artisan can readily identify such regionsin a virus by, for example, randomly testing segments of virus DNA forregions that allow recombinant formation without seriously affectingvirus viability of the recombinant. One region that can readily be usedand is present in many viruses is the thymidine kinase (TK) gene. Forexample, the TK gene has been found in all pox virus genomes examined[leporipoxvirus: Upton et al, J. Virology, 60:920 (1986) (shope fibromavirus); capripoxvirus: Gershon et al, J. Gen. Virol., 70:525 (1989)(Kenya sheep-1); orthopoxvirus: Weir et al, J. Virol., 46:530 (1983)(vaccinia); Esposito et al, Virology, 135:561 (1984) (monkeypox andvariola virus); Hruby et al, PNAS, 80:3411 (1983) (vaccinia); Kilpatricket al, Virology, 143:399 (1985) (Yaba monkey tumor virus); avipoxvirus:Binns et al, J. Gen. Virol. 69:1275 (1988) (fowlpox); Boyle et al,Virology, 156:355 (1987) (fowlpox); Schnitzlein et al, J. VirologicalMethods, 20:341 (1988) (fowlpox, quailpox); entomopox (Lytvyn et al, J.Gen. Virol., 73:3235-3240 (1992)].

In vaccinia, in addition to the TK region, other insertion regionsinclude, for example, the Hind III M fragment.

In fowlpox, in addition to the TK region, other insertion regionsinclude, for example, the BamHI J fragment [Jenkins et al, AIDS Researchand Human Retroviruses 7:991-998 (1991)] the EcoRI-HindIII fragment,EcoRV-HindIII fragment, BamHI fragment and the HindIII fragment setforth in EPO Application No. 0 308 220 A1. [Calvert et al, J. of Virol.67:3069-3076 (1993); Taylor et al, Vaccine 6:497-503 (1988); Spehner etal (1990) and Boursnell et al, J. of Gen. Virol. 71:621-628 (1990)].

In swinepox preferred insertion sites include the thymidine kinase generegion.

In addition to the requirement that the gene be inserted into aninsertion region, successful expression of the inserted gene by themodified poxvirus requires the presence of a promoter operably linked tothe desired gene, i.e. in the proper relationship to the inserted gene.The promoter must be placed so that it is located upstream from the geneto be expressed. Promoters are well known in the art and can readily beselected depending on the host and the cell type you wish to target. Forexample in poxviruses, pox viral promoters should be used, such as thevaccinia 7.5 k, 40K or fowlpox promoters such as FPV C1A. Enhancerelements can also be used in combination to increase the level ofexpression. Furthermore, the use of inducible promoters, which are alsowell known in the art, in some embodiments are preferred.

A specific immune response for PSA can be generated by administeringbetween about 10⁵-10⁹ pfu of the recombinant pox virus, constructed asdiscussed above to a host, more preferably one uses 10⁷ pfu. Thepreferred host is a human. At least one interval thereafter, which ispreferably one to three months later, the immune response is boosted byadministering additional antigen to the host. More preferably there isat least a second “boost” preferably one to three months after the firstboost. The antigen may be administered using the same pox virus vector.The antigen may preferably be administered using a second pox virusvector from a different pox genera, or may be administered directlyusing, for example, an adjuvant or liposome. Cytokines, e.g. IL-2, IL-6,IL-12 or co-stimulatory molecules, e.g. B7.1, B7.2 may be used asbiologic adjuvants and can be administered systemically to the host orco-administered via insertion of the genes encoding the molecules intothe recombinant pox vector.

Adjuvants include, for example, RIBI Detox (Ribi Immunochemical), QS21and incomplete Freund's adjuvant.

Generation of Cytotoxic T-Cells

Cytotoxic T-cells specific for PSA can be established from peripheralblood mononuclear cells (PBMC) obtained from a host immunized asdiscussed above. For example, PBMC can be separated by using LymphocyteSeparation Medium gradient (Organon Teknika, Durham, N.C., USA) aspreviously described [Boyum et al, Scand J. Clin Lab Invest 21:77-80(1968)]. Washed PBMC are resuspended in a complete medium, for example,RPMI 1640 (GIBCO) supplemented with 10% pool human AB serum (Pel-FreezeClinical System, Brown Dear, Wis., USA), 2 mM glutamine, 100 U/mlpenicillin and 100 μg/ml of streptomycin (GIBCO). PBMC at aconcentration of about 2×10⁵ cells in complete medium in a volume of,for example 100 μl are added into each well of a 96-well flat-bottomassay plate (Costar, Cambridge, Mass., USA). The antigen or peptides areadded into the cultures in a final concentration of about 50 μg/ml andincubated at 37° C. in a humidified atmosphere containing 5% CO₂ for 5days. After removal of peptide containing media, the cultures areprovided with fresh human IL-2 (10 U/ml) after 5 days and replenishedwith IL-2 containing medium every 3 days. Primary cultures arerestimulated with the same peptide (50 μg/ml) on day 16. 5×10⁵irradiated (4,000 rad) autologous PBMC are added in a volume of about 50μl complete medium as antigen-presenting cells (APC). About five dayslater, the cultures are provided with human IL-2 containing medium asdescribed previously. Cells are restimulated for 5 days at intervals of16 days.

Epitope Mapping

The cytotoxic T-cells of the present invention can be used to determinethe epitope of the PSA that elicits a cytotoxic T-cell. For example, onecan cut the PSA into numerous peptide fragments. Alternatively, thefragments can be chemically synthesized. Cytotoxic T-cells can then beplated and different fragments added to different wells. Only T-cellswhich recognize one of the pre-selected peptide fragments as an epitopewill continue to expand, thereby permitting ready identification.

These fragments can then be used to elicit cytotoxic T-cell instead ofusing the whole protein. Additionally, one can prepare other fragmentscontaining the epitope to enhance its ability to elicit a cytotoxicT-cell response.

Modifications to these fragments are well known in the art and includethe use of conjugates, specific amino acid residues such as cystines,etc.

Drug Assay

The cytotoxic T-cell can also be used to screen for compounds whichenhance the ability of the antigen to create a cytotoxic T-cellresponse. For example, cytotoxic T-cells can be incubated with aselected epitope, for example, in a microtiter plate. The compound to betested, e.g. a drug, is then added to the well and the growth of theT-cells is measured. T-cell expansion indicates that the test compoundenhances the T-cell response. Such compounds can be further evaluated.

Therapy

The cytotoxic T-cell can be cultured to amplify its number and theninjected back into the host by a variety of means. Generally, between1×10⁵ and 2×10¹¹ cytotoxic T-cells per infusion are administered in, forexample, one to three infusions of 200 to 250 ml each over a period of30 to 60 minutes. After the completion of the infusions, the patient maybe treated with recombinant interleukin-2 with a dose of 720,000 IU perkilogram of body weight intravenously every eight hours; some doses canbe omitted depending on the patient's tolerance for the drug. Inaddition, after infusion, additional antigen or fragments containingT-cell eliciting epitope(s) may be administered to the patient tofurther expand the T-cell number. The antigen or epitope may beformulated with an adjuvant and/or may be in a liposomal formulation.

The cytotoxic T-cells can also be modified by introduction of a viralvector containing a DNA encoding TNF and reintroduced into a host in aneffort to enhance the anti-tumor activity of the cells. Other cytokinescan also be used.

The recombinant vector can be administered using any acceptable route,including, for example, scarification and injection, e.g. intradermal,subcutaneous, intramuscular, intravenous or intraperitoneal.

For parenteral administration, the recombinant vectors will typically beinjected in a sterile aqueous or non-aqueous solution, suspension oremulsion in association with a pharmaceutically-acceptable carrier suchas physiological saline.

Reference Example 1 Construction of Vectors Pox Viruses

A number of pox viruses have been developed as live viral vectors forthe expression of heterologous proteins (Cepko et al, Cell 37:1053-1062(1984); Morin et al, Proc. Natl. Acad. Sci. USA 84:4626-4630 (1987);Lowe et al, Proc. Natl. Acad. Sci. USA, 84:3896-3900 (1987); Panicali &Paoletti, Proc. Natl. Acad. Sci. USA, 79:4927-4931 (1982); Mackett etal, Proc. Natl. Acad. Sci. USA. 79:7415-7419 (1982)). Representativefowlpox and swinepox virus are available through the ATCC underaccession numbers VR-229 and VR-363, respectively.

DNA Vectors for In Vivo Recombination with a Parent Virus

Genes that code for desired carcinoma associated antigens are insertedinto the genome of a pox virus in such a manner as to allow them to beexpressed by that virus along with the expression of the normalcomplement of parent virus proteins. This can be accomplished by firstconstructing a DNA donor vector for in vivo recombination with a poxvirus.

In general, the DNA donor vector contains the following elements:

-   -   (i) a prokaryotic origin of replication, so that the vector may        be amplified in a prokaryotic host;    -   (ii) a gene encoding a marker which allows selection of        prokaryotic host cells that contain the vector (e.g. a gene        encoding antibiotic resistance);    -   (iii) at least one gene encoding a desired protein located        adjacent to a transcriptional promoter capable of directing the        expression of the gene; and    -   (iv) DNA sequences homologous to the region of the parent virus        genome where the foreign gene(s) will be inserted, flanking the        construct of element (iii).

Methods for constructing donor plasmids for the introduction of multipleforeign genes into pox virus are described in WO91/19803, the techniquesof which are incorporated herein by reference. In general, all DNAfragments for construction of the donor vector, including fragmentscontaining transcriptional promoters and fragments containing sequenceshomologous to the region of the parent virus genome into which foreigngenes are to be inserted, can be obtained from genomic DNA or cloned DNAfragments. The donor plasmids can be mono-, di-, or multivalent (i.e.can contain one or more inserted foreign gene sequences).

The donor vector preferably contains an additional gene which encodes amarker which will allow identification of recombinant viruses containinginserted foreign DNA. Several types of marker genes can be used topermit the identification and isolation of recombinant viruses. Theseinclude genes that encode antibiotic or chemical resistance (e.g. seeSyropoulos et al, J. Virol., 62:1046 (1988); Falkner and Moss, J.Virol., 62:1849 (1988); Franke et al, Mol. Cell. Biol., 5:1918 (1985),as well as genes such as the E. coli lacZ gene, that permitidentification of recombinant viral plaques by calorimetric assay(Panicali et al, Gene, 47:193-199 (1986)).

Integration of Foreign DNA Sequences into the Viral Genome and Isolationof Recombinants

Homologous recombination between donor plasmid DNA and viral DNA in aninfected cell results in the formation of recombinant viruses thatincorporate the desired elements. Appropriate host cells for in vivorecombination are generally eukaryotic cells that can be infected by thevirus and transfected by the plasmid vector. Examples of such cellssuitable for use with a pox virus are chick embryo fibroblasts, HuTK143(human) cells, and CV-1, Vero and BSC-40 (monkey) cells. Infection ofcells with pox virus and transfection of these cells with plasmidvectors is accomplished by techniques standard in the art (Panicali andPaoletti, U.S. Pat. No. 4,603,112, WO89/03429).

Following in vivo recombination, recombinant viral progeny can beidentified by one of several techniques. For example, if the DNA donorvector is designed to insert foreign genes into the parent virusthymidine kinase (TK) gene, viruses containing integrated DNA will beTK- and can be selected on this basis (Mackette et al, Proc. Natl. Acad.Sci. USA 79:7415 (1982)). Alternatively, co-integration of a geneencoding a marker or indicator gene with the foreign gene(s) ofinterest, as described above, can be used to identify recombinantprogeny. One preferred indicator gene is the E. coli lacZ gene:recombinant viruses expressing 6-galactosidase can be selected using achromogenic substrate for the enzyme (Panicali et al, Gene, 47:193(1986)).

Characterizing the Viral Antigens Expressed by Recombinant Viruses

Once a recombinant virus has been identified, a variety of methods canbe used to assay the expression of the polypeptide encoded by theinserted gene. These methods include black plaque assay (an in situenzyme immunoassay performed on viral plaques), Western blot analysis,radioimmunoprecipitation (RIPA), and enzyme immunoassay (EIA).

Example I Generation of PSA Specific Immune Response Materials andMethods

Recombinant Vaccinia Virus

A 786 bp DNA fragment encoding the entire open reading frame of humanprostate specific antigen was amplified by reverse transcriptase PCR(GeneAmp RNA PCR Kit, Perkin Elmer, Norwalk, Conn.) from total RNAextracted from the human metastatic prostate adenocarcinoma cell line,LNCaP.FGC (CRL 1740, American Type Culture Collection (ATCC), Rockville,Md.). The predicted amino acid sequence derived from the PSA codingsequence was shown to be nearly identical to the published sequence(Lundwall et al, 1987), differing only in a change from asparagine totyrosine at position 220. The PSA DNA fragment, containing the entirecoding sequence for PSA, 41 nucleotides of the 5′ untranslated region,and 520 nucleotides of the 3′ untranslated region, was inserted into theXba 1 restriction endonuclease cleavage site of the vaccinia virustransfer vector pT116. The resulting plasmid, designated pT1001,contains the PSA gene under the control of the vaccinia virus 40Kpromoter (Gritz et al 1990) and the E. coli lacZ gene under the controlof the fowlpox virus C1 promoter (Jenkins et al, 1991). The foreigngenes are flanked by DNA sequences from the Hind III M region of thevaccinia genome. A plaque-purified isolate from the Wyeth (New York CityBoard of Health) strain of vaccinia was used as the parental virus inthe construction of the recombinant vaccinia virus. The generation ofrecombinant vaccinia virus was accomplished via homologous recombinationbetween vaccinia sequences in the Wyeth vaccinia genome and thecorresponding sequences in pT1001 in vaccinia-infected RK₁₃ cells (CCL37, ATCC) transfected with pT1001. Other cell lines for generation ofthe recombinant include but are not limited to CV-1 and Vero.Recombinant virus was identified using a chromogenic assay, performed onviral plaques in situ, that detects expression of the lacZ gene productin the presence of halogenated indolyl-beta-D-galactoside (Bluo-gal), asdescribed previously (Panicali et al, 1986). Appropriate bluerecombinant viruses were purified by four rounds of plaque-purification.Virus stocks were prepared by clarifying infected RK₁₃ cell lysatesfollowed by centrifugation through a 36% sucrose cushion.

Characterization of Recombinant Virus Southern Analysis of DNARecombination

The recombinant vaccinia genome was analyzed by viral DNA extraction,restriction endonuclease digestion with Hind III and Southern blottingas previously described (Kaufman et al, 1991).

Western Analysis of Protein Expression

Confluent BSC-40 cells were infected with either parental wild typevaccinia virus (designated V-Wyeth) or recombinant vaccinia-PSA(designated rV-PSA) at an MOI of 1 in Dulbecco's Modified Eagle's Mediumcontaining 2% fetal bovine serum. After an overnight infection, themedium was removed from the cells, and an aliquot was methanolprecipitated to assay for the presence of secreted PSA. The infectedcells were lysed in hypotonic lysis buffer (150 mM NaCl, 0.05% EDTA, 10mM KCl, 1 mM PMSF) and then sonicated. Cell lysates and culture mediawere electrophoresed on an SDS-10% acrylamide gel. The proteins weretransblotted to nitrocellulose and the blot was incubated with a rabbitantibody specific for PSA (P0798, Sigma Chemical Co., St. Louis, Mo.)for 4 hours at ambient temperature, washed and then incubated with goatanti-rabbit phosphatase-labeled secondary antibody (AP, Kirkegaard &Perry Laboratories, Gaithersburg, Md.) and developed according to themanufacture's instructions.

Generation of B-Cell Lines

Monkey autologous B lymphoblastoid cell lines (BLCL) were established byinfecting 1×10⁵ freshly isolated PBMCs in 100 ml of RPMI 1640supplemented with L-glutamine, gentamicin, and 10% FCS (Biofluids,Rockville, Md.) with 100 ml supernatant from S594 cells (kindly providedby Dr. M.D. Miller, Harvard Medical School, New England Regional PrimateResearch Center, Southborough, Mass.) which contains the baboonherpesvirus Herpes papio, in a 96 well, flat-bottomed plate (Costar,Cambridge, Mass.). Following transformation, cells were expanded andmedia changed once weekly.

Immunization of Monkeys

Twelve juvenile male rhesus monkeys (Macaca mulatta), ages 1 to 2 years,were assigned to three vaccination groups of four animals each. Oneanimal from each group was prostatectomized. Animals were immunized 3times on days 1, 29 and 57. Doses of either 1×10⁷ or 1×10⁸ PFU of rV-PSAwere administered to 4 animals by skin scarification. V-Wyeth (1×10⁸PFU) was administered to 4 animals as controls. The animals were housedand maintained at the Toxicology Research Laboratory, University ofIllinois at Chicago (TRL/UIC) in accordance with the guidelines of theNational Cancer Institute Animal Care and Use Committee and the Guidefor the Care and Use of Laboratory Animals (Department of Health andHuman Services Publication NIH 85-23, revised 1985 by the FDA Center forBiologics Evaluation and Research Office of Biological Product Review,Division of Product Quality Control, Pathology and PrimatologyLaboratory, Bethesda, Md.).

Toxicology

Physical examinations were performed on ketamine (Ketamine® HCl, 10mg/kg 1.M.) sedated animals. Rectal temperatures and weights wererecorded for each monkey on a weekly basis. The vaccination site wasobserved and erythema and swelling were measured by caliper. Each animalwas examined for regional lymphadenopathy, hepatomegaly, andsplenomegaly. Any other gross abnormalities were also recorded.

Blood was obtained by venipuncture from the femoral vein of ketaminesedated animals before and after each immunization. A complete bloodcount, differential hepatic and renal chemistry evaluation was performedon each monkey by TRL/UIC. Results were compared to normal primatevalues (Kantor et al., 1992b). Circulating levels of PSA before andafter immunization were analyzed by radioimmunoassay (Tandem™,Hybritech, San Diego, Calif.).

Measurement of Antibody Titers

Prior to each immunization and 2 weeks following each immunization,anti-PSA antibody was quantified by ELISA. Microtiter plates were coatedwith purified PSA (100 ng/well, Calbiochem, La Jolla, Calif.), ovalbumin(100 ng/well, Sigma), or 1×10⁷ PFU/well UV-inactivated V-Wyeth in PBS.The plates were blocked with 2% BSA in PBS, dried and stored at −20° C.until used. The plates were incubated with serum diluted 1:5, as well asa monoclonal antibody for PSA (DAKO M750, Denmark) as a standardcontrol, for 24 hours at 4° C. Plates were washed several times with PBScontaining 1% BSA, and incubated at 37° C. for 45 min with horseradishperoxidase-conjugated goat anti-human IgG or IgM heavy chain specificantiserum (1:8000) (Southern Biotechnology Associates, Birmingham, Ala.)and antibody detected by HRP substrate system (Kirkegaard & PerryLaboratories, Gaithersburg, Md.) according to the manufacture'sinstructions. The absorbance of each well was read at 405 nm using aBio-Tek EL310 microplate ELISA reader (Winooski, Vt.).

Lymphoproliferative Assay

Autologous monkey BLCL were plated at a density of 3×10⁶ cells/wells in24 well plates with 160 mg/well purified PSA (Fitzgerald, Concord,Mass.) or 160 mg/well ovalbumin (Sigma) a 37° C. for 24 hours. Cellswere then γ-irradiated (14000 rad), harvested, washed and suspended at afinal concentration of 1×10⁷/ml. Fresh monkey PBMCs from heparinizedblood, 6 weeks to 7 months after the last immunization, were isolated onlymphocyte separation medium (Organon Teknika, West Chester, Pa.).Lymphoproliferative responses were evaluated by co-culturing 1.5×10⁵cells with 5×10⁵ cells/well of autologous BLCL in 0.2 ml of RMPI 1640supplemented with 10% heat-inactivated fetal calf serum in flat-bottomed96 well plates (Costar) for 5 days. PBMCs were cultured with 2×10⁷PFU/ml UV-irradiated V-Wyeth as a recall antigen or 2 mg/ml Con-A aspositive controls. Cells were labeled for the final 12-18 h of theincubation with 1 mCi/well [³H]thymidine (New England Nuclear,Wilmington, Del.) and harvested with a PHD cell harvester (CambridgeTechnology, Cambridge, Mass.). The incorporated radioactivity wasmeasured by liquid scintillation counting (LS 6000IC; Beckman, Duarte,Calif.). The results from triplicate wells were averaged and arereported as mean±SEM.

Results Generation and Characterization of Recombinant Virus

The cDNA fragment encoding the open reading frame of human PSA wasobtained by reverse transcriptase PCR using PSA specific oligonucleotideprimers 5′ TCTAGAAGCCCCAAGCTTACCACCTGCA 3′ (SEQ. ID. NO.: 16), 5′TCTAGATCAGGGGTTGGCCACGATGGTGTCCTTGATCCACT 3′ (SEQ. ID. NO.: 17), andligated into the vaccinia virus transfer vector pT106. This vectorcontains a strong vaccinia virus early/late promoter (designated P40)upstream of the multiple cloning sited to drive the synthesis of theinserted gene product. The ligation and orientation of the PSA DNAfragment, as well as promoter position were verified by PCR andsequencing. The chimeric vector construct was inserted into the vaccinevirus genome Hind III M site by homologous recombination as previouslyreported (Kaufman, et al., (1991)), and confirmed by Southern analysisprobing with ³²P radiolabeled DNA corresponding to PSA sequences andvaccinia sequences in the Hind III M region (data not shown). The entirecDNA sequence of PSA in the vaccinia virus clone was shown to be nearlyidentical to the published sequences (Lundwall, et al., 1987).

Expression of recombinant protein was confirmed by western blot analysisof supernatant fluids and protein extracts from rV-PSA infected BSC-40cells. These cells are routinely used for the evaluation of recombinantvaccinia products (Moss, et al., 1993). Incubation of cell supernatantblots from rV-PSA infected cells with rabbit anti-PSA antibody revealeda single immunoreactive polypeptide of approximately 33,000 daltons(FIG. 1, lanes 2-4). Similarly, incubation of protein extract blots fromrV-PSA infected cells revealed a single band of the same molecularweight (FIG. 1, lanes 7-9). This is consistent with the predicted sizeof the PSA molecule (Armbruster, et al., 1993; Wang, et al., 1982). Cellsupernatant blots (lane 1) or protein extract blots (lane 6) from cellsinfected with parental strain V-Wyeth remained negative for expressionof PSA. These results thus demonstrate that a recombinant vaccinia viruscan faithfully express the human PSA gene product.

Rhesus Monkey Model

The prostate gland of the rhesus monkey is structurally and functionallysimilar to the human prostate (Wakui, et al., 1992). At the molecularlevel, there is 94% homology between both the amino acid and nucleicacid sequences of rhesus PSA (Gauthier, et al., 1993) and human prostatespecific antigen (Kerr, et al., 1995; Lundwall, et al., 1987). Human PSAis essentially an autoantigen in the rhesus monkey.

Experimental Design

Table 1 delineates the protocol used in the immunization of 12 rhesusmonkeys with either rV-PSA or the control V-Wyeth by skin scarification.Three groups of 4 animals were immunized with either rV-PSA at 1×10⁷PFU/dose, rV-PSA at 1×10⁸ PFU/dose, or V-Wyeth at 10⁸ PFU/dose 3 timesat 4 week intervals. These doses were chosen to ascertain the maximumtolerated dose for safety as well as to obtain maximum humoral andcell-mediated responses to PSA.

The rhesus monkeys were divided into 3 groups: high dose V—Wyeth, lowdose rV-PSA, and high-dose rV-PSA. One animal in each group wassurgically prostatectomized to parallel two situations with regard topotential therapy in humans: (a) prostate intact, with primary and/ormetastatic disease; or (b) patients prostatectomized with prostatecancer metastatic deposits. The presence of an intact prostate glandcould conceivably serve as an antigen ‘sink’, either inducing anergythrough persistence of antigen, or masking immunological effects bysequestering reactive cells or antibodies.

Physical Consequence of Immunization

The area of the lesions induced by rV-PSA or V-Wyeth was analyzed 7 daysfollowing each inoculation. In general, more induration was seen afterthe first inoculation, compared to the second inoculation (FIG. 2A).After the third inoculation, there was no swelling of the vaccinationsite. The duration of the lesion following each immunization was shorterafter each inoculation (FIG. 2B). Regional lymph node swelling followingvaccination was greater in most monkeys following the firstimmunization, compared to the second, or third immunization (FIG. 2C).In general, no differences were seen in these parameters with the use ofrV-PSA or V-Wyeth. Monkeys receiving V-Wyeth were compared with thosereceiving rV-PSA with respect to constitutional symptoms. Mildtemperature elevations were seen in all animals following vaccination.There was no evidence of weight loss, hepatomegaly or splenomegaly inany of the animals, and there was no differences between V-Wyeth orrV-PSA treated animals (data not shown). Animals were tested forcomplete blood count, differential, and hepatic and renal chemistries.Complete blood counts remained within normal limits throughout the studyin both V-Wyeth and rV-PSA immunized animals (Table 2). Hepatic andrenal functions were assessed prior to immunization and 12 weeksfollowing primary immunization (Table 3). Parameters analyzed includedalkaline phosphatase, blood urea nitrogen, alanine aminotransferase,aspartate aminotransferase, lactate dehydrogenase, and creatine andcreatine kinase levels. There was no significant difference betweenanimals receiving V-Wyeth or rV-PSA. There was no detectable PSA in thecirculation of any of these monkeys after any immunization (detectionlimit was 0.1 ng/ml). At this time, which is 54 weeks post allimmunizations, no toxicities were observed in monkeys of any of thegroups, including those which were prostatectomized.

PSA Specific Humoral Responses

As indicated in Table 1, monkeys 1-4 were administered V-Wyeth whilemonkeys 5-12 were administered rV-PSA. Sera from each of these monkeyswere analyzed by ELISA for immunoreactivity to PSA or UV-inactivatedV-Wyeth, and ovalbumin as control antigen. Sera obtained from monkeysprior to vaccination were negative for reactivity to PSA (Table 4, PI).Fifteen days following primary immunization, monkeys in both the 1×10⁸and 1×10⁷ dose rV-PSA groups developed low titer IgM antibodies specificfor PSA (titers were determined at a 1:5 serum dilution). Although otherisotypes of antibody were analyzed (IgG, IgA, IgM), only IgM was inducedby rV-PSA throughout the observation period of 270 days. The antibodytiters decreased over the 4 weeks prior to the next inoculation. Priorto the second vaccination on day 29, 3 of 4 animals in the 1×10⁷rV-PSAgroup remained positive for PSA antibody, while 4 of 4 animals remainedpositive in the 1×10⁸rV-PSA group. Anti-PSA antibody titers increasedafter the second vaccination on day 29, but remained static after thethird vaccination on day 57. By 270 days after the primary immunization,all animals were negative for PSA IgM antibody. Monkeys remainednegative for IgG specific for PSA throughout the observation period(data not shown). There was no correlation between rV-PSA dose andanti-PSA IgM titer, nor was there any apparent effect of prostatectomy.All monkey sera were negative for IgG or IgM to ovalbumin at all timepoints; as a positive control, however, the IgG titer in all threetreatment groups to vaccinia virus was greater than 1:2000 as early as29 days after the primary immunization (data not shown).

In general, vaccinia virus is a weak human pathogen (Paoletti et al.,1993). Following vaccination, local erythema, induration, low-gradefever, and regional lymphadenopathy are common. The virus replicates inthe epidermal cells of the skin and the virus is usually cleared within14 days. All monkeys, whether given V-Wyeth or rV-PSA, exhibited theusual low grade constitutional symptoms of a vaccinia virus infection(FIG. 2). There was no evidence of any adverse effects as indicated bychanges in blood counts, differentials, hepatic and renal chemistries(Tables 2-3). The monkeys appeared healthy, without any physical signsof toxicity, throughout the 54 weeks of observation.

Although the rV-PSA construct was unable to elicit an anti-PSA IgGresponse, PSA specific IgM responses were noted in all rV-PSA immunizedmonkeys regardless of dose level (Table 4). These antibody responseswere of low titer, short lived and could not be boosted, indicatinginduction of a primary response but not memory B-cells or affinitymaturation.

PSA Specific Lymphoproliferative Assay

PSA specific T-cell responses in monkeys immunized with rV-PSA orV-Wyeth were analyzed using a lymphoproliferative assay. As seen inTable 5, the PBMCs from all monkeys analyzed responded, regardless ofwhether they received rV-PSA or V-Wyeth, to the lymphocyte mitogenconcanavalin-A, as well as with the recall antigen UV-inactivatedV-Wyeth. Differential responses to PSA versus medium alone or ovalbuminwere seen in 1 animal (number 6) in the 1×10⁷ PFU rV-PSA group. AllPBMCs from animals in the 1×10⁸ PFU rV-PSA group, however, responded toPSA in this assay. This experiment was repeated 5 times with similarresults and data shown in Table 5 is from PBMCs isolated from monkeys270 days after the primary immunization. No differences in PSA specificT-cell responses were seen in the prostatectomized monkeys.

To investigate cell mediated responses to the administration of rV-PSA,lymphoproliferative assays were performed using PBMCs from animalsreceiving the recombinant vaccine. One of four monkeys receiving thelower dose of rV-PSA (1×10⁷ PFU) and four of four receiving the higherdose (1×10⁸ PFU) maintained specific T-cell responses to PSA protein upto 270 days following primary immunization as indicated by thelymphoproliferative assay (Table 5). Prostatectomy appeared to have noeffect on either the humoral or cellular responses of monkeys receivingrV-PSA. Evidence of PSA specific T-cell responses in monkeys lackingmature antibody isotypes could be due to two distinct events followingvaccination with rV-PSA: a T-cell independent event, leading to IgMproduction, and a T-cell dependent event, leading to specificlymphoproliferative responses.

TABLE 1 Inoculation protocol of rhesus monkeys with the PSA recombinantand wild-type vaccinia virus Dose* Monkey Prostate Immunogen (PFU) 1 YesV-Wyeth 1 × 10⁸ 2 Yes V-Wyeth 1 × 10⁸ 3 Yes V-Wyeth 1 × 10⁸ 4 No V-Wyeth1 × 10⁸ 5 Yes rV-PSA 1 × 10⁷ 6 Yes rV-PSA 1 × 10⁷ 7 Yes rV-PSA 1 × 10⁷ 8No rV-PSA 1 × 10⁷ 9 Yes rV-PSA 1 × 10⁸ 10 Yes rV-PSA 1 × 10⁸ 11 YesrV-PSA 1 × 10⁸ 12 No rV-PSA 1 × 10⁸ *All animals received 3immunizations at 4 week intervals.

TABLE 2 Mean WBC count, hematocrit, and differential count in rhesusmonkeys receiving recombinant or wild-type vaccine V-Wyeth (n = 4)rV-PSA (n = 8) Before After Before After Test Normal rangesimmunization^(a) immunization^(b) immunization immunization WBC 7-15 ×10³ 5.0 ± 0.8 5.1 ± 0.5 5.2 ± 0.7 5.8 ± 0.9 Hematocrit 33-43 37.4 ± 0.2 37.0 ± 0.1  37.8 ± 0.4  37.0 ± 0.5  (vol. %) Lymphocytes  1-7 × 10³ 2.8± 0.7 3.9 ± 0.5 2.2 ± 0.4 3.5 ± 0.8 SEGS^(c) (%)  3-69 2.0 ± 0.2 0.78 ±0.2  2.9 ± 0.6 1.9 ± 0.3 Monocytes (%) 0-8  0.1 ± 0.05  0.2 ± 0.04  0.1± 0.04  0.2 ± 0.50 Eosinophils 0-8  0.1 ± 0.02  0.2 ± 0.10  0.1 ± 0.03 0.1 ± 0.02 (%) ^(a)1 week prior to primary immunization ^(b)12 weeksfollowing primary immunization ^(c)Segmented lymphocytes

TABLE 3 Mean serum chemistry values in rhesus monkeys receivingrecombinant or wild-type vaccine. V-Wyeth (n = 4) rV-PSA (n = 8) BeforeAfter Before After Test Normal ranges immunization^(a) immunization^(b)immunization immunization ALKP^(c) (u/l) 200-800 451 ± 48 610 ± 33 339 ±74 454 ± 47 BUN^(d) (mg/dl) 12-30 19.0 ± 3.0 17.8 ± 0.9 17.1 ± 0.6 20.5± 1.0 ALT^(e) (u/l) 20-60 25.2 ± 1.9 22.8 ± 1.0 28.9 ± 5.3 25.8 ± 1.6AST^(f) (u/l) 40-80 37.8 ± 2.3 31.8 ± 4.4 37.9 ± 3.6 31.9 ± 2.4 LDH^(g)(u/l) 200-500 194 ± 20 212 ± 21 236 ± 41 194 ± 13 Creatine 0.5 ± 1.0 0.9 ± 0.10  0.8 ± 0.03  0.8 ± 0.05  0.8 ± 0.02 (mg/dl) Creatine 500-2000  662 ± 112  466 ± 119  498 ± 120 563 ± 81 Kinase (u/l) ^(a)1week prior to primary immunization ^(b)12 weeks following primaryimmunization ^(c)Alkaline phosphatase ^(d)Blood urea nitrogen^(e)Alanine aminotransferase ^(f)Asparate aminotransferase ^(g)Lactatedehydrogenase

TABLE 4 Primate IgM^(a) Response to Inoculation with rV-PSA Dose DaysPost Immunization^(b) Monkey Immunogen (PFU) PI^(d) 15 29^(e) 43 57^(e)71 270 1 V-Wyeth 1 × 10⁸  ND^(f) ND ND ND ND ND ND 2 V-Wyeth 1 × 10⁸ NDND ND ND ND ND ND 3 V-Wyeth 1 × 10⁸ ND ND ND ND ND ND ND  4^(c) V-Wyeth1 × 10⁸ ND ND ND ND ND ND ND 5 rV-PSA 1 × 10⁷ ND >40 5 20 >40 >40 ND 6rV-PSA 1 × 10⁷ ND >40 ND ND 20 20 ND 7 rV-PSA 1 × 10⁷ ND >40 5 20 20 20ND  8^(c) rV-PSA 1 × 10⁷ ND >40 5 10 >40 >40 ND 9 rV-PSA 1 × 10⁸ ND 20 520 10 10 ND 10  rV-PSA 1 × 10⁸ ND 20 5 40 >40 NT^(g) ND 11  rV-PSA 1 ×10⁸ ND >40 >40 >40 >40 >40 ND 12^(c ) rV-PSA 1 × 10⁸ ND >40 20 40 20 20ND ^(a)All monkeys seras were negative for IgG to PSA at all timepoints; All seras were positive for IgG to vaccinia virus (>1:2000) atday 71. ^(b)Monkeys received vaccinations on days 1, 29 and 57. Sera(1:5) was tested by ELISA. Titers were calculated using an O.D. of 0.4.^(c)Animal was prostatectomized. ^(d)PI, Pre-immune. ^(e)Animals bledbefore boosting. ^(f)ND, not detectable; limit of detection was <1:5dilution. ^(g) NT, not tested.

TABLE 5 PSA Specific Lymphoproliferative T-cell Responses of RhesusPBMCs 270 Days Following Inoculation with rV-PSA Antigen^(a) Mon- Immu-Dose UV- key nogen (PFU) Medium Con A Oval Wyeth PSA^(d) 1 V-Wyeth 1 ×10⁸ 397 65701 376 24785 414  2^(b) V-Wyeth 1 × 10⁸ NT NT NT NT NT 3V-Wyeth 1 × 10⁸ 450 84860 522 18859 413  4^(c) V-Wyeth 1 × 10⁸ 532107840 553 16571 387 5 rV-PSA 1 × 10⁷ 412 85276 408 6040 539 6 rV-PSA 1× 10⁷ 401 96368 404 7776 3,134 7 rV-PSA 1 × 10⁷ 417 90801 522 10908 434 8^(c) rV-PSA 1 × 10⁷ 1069 99216 744 15346 484 9 rV-PSA 1 × 10⁸ 384106248 386 14499 10,635 10  rV-PSA 1 × 10⁸ 432 92263 404 19872 18,56111  rV-PSA 1 × 10⁸ 411 94055 1063 5124 16,245 12^(c ) rV-PSA 1 × 10⁸ 420124896 392 11944 12,945 ^(a)Antigen concentrations were: Con a (2μg/ml); Ovalbumin (100 μg/ml); UV Wyeth (2 × 10⁷ pfu/ml); and PSA (100μg/ml). Each value represents a mean CPM of triplicate samples. Standarddeviation never exceeded 10%. ^(b)NT, not tested. B-cells were nottransformed for this animal. ^(c)Animal was prostatectomized. ^(d)Valuesin bold are significant when compared to their respective medium controlvalues (p < 0.001).

Example II Identification of Potential Prostate Specific Antigen (PSA)Specific T Cell Epitopes

Since the entire amino acid sequence of human PSA is known and humanclass I HLA A2 consensus motifs have been described, studies wereundertaken to identify a series of peptides that would potentially bindclass I A2 molecules. A2 was chosen since it is most common HLA class Imolecule being represented in approximately 50% of North AmericanCaucasians and 34% of African Americans. The peptide sequence of PSA wasthus examined for matches to the consensus motifs for HLA A2 bindingpeptides. Peptides were only selected if their sequence divergedsufficiently from the PSA-related human glandular kallikrein (HGK) geneand pancreatic kallikrein antigen (PKA) sequences.

The amino acid sequence of human PSA was scanned using a predictivealgorithm that combines a search for anchor residues with numericalassignments to all residues at all positions. The T2 cell binding assaywas then used to determine which peptides bound human HLA A2 molecules.As can be seen in Table 6, PSA peptides 141-150, 154-163 and 146-154scored positive in this assay (Nijman, H. W., et al., Eur. J. Immunol.23:1215-1219, 1993). Table 7 gives the amino acid sequence of thesepeptides and compares them to corresponding sequences of HGK and PKA.

TABLE 6 PSA peptide binding assay Antigen MAb A2, 69 None 127.25^(a) PSA141-150 230.34 PSA 146-154 223.97 PSA 154-163 182.30 Peptides were usedat a concentration of 50 μg/ml ^(a)Mean channel fluorescent intensity.CIRA2 cell line was used as positive control for anti-A2 staining[(241.15)].

TABLE 7 PSA peptide amino acid sequence PSA 141-150 F L T P K K L Q C V(Seq. ID No. 1) HGK - - R - R S - - - - (Seq. ID No. 7) PKA - S F - DD - - - - (Seq. ID No. 8) PSA 146-154 K L Q C V D L H V (Seq. ID No. 9)HOK S - - - - S - - L (Seq. ID No. 10) PKA D - - - - - - K I (Seq. IDNo. 11) PSA 154-163 V I S N D V C A Q V (Seq. ID No. 2) HGK L L - - -M - - R A (Seq. ID No. 12) PKA I L P - - E - E K A (Seq. ID No. 13)

Example III Establishment of Human T Cell Lines Cytolytic For HumanTumor Cells Expressing PSA

PBMC from normal healthy donors expressing the HLA A2 class 1 allelewere used in an attempt to determine if PSA specific peptides areimmunogenic for humans. Peptides 141-150 and 154-163 were used in thisstudy. The methodology used for the establishment of these cell linesinvolves pulsing of PBMC with peptide and IL-2 as previously described(Tsang, K. Y., et al, 1995, J. Nat'l Cancer Inst., Vol. 87(13):982-90and in U.S. application Ser. No. 08/396,385, the disclosure of which isherein incorporated by reference). T cell lines were able to beestablished from 5/6 normal donors using PSA peptide 141-150 and from6/6 normal donors using PSA peptide 154-163. Moreover, PBMC wereobtained from two prostate cancer patients. T cell lines wereestablished from these PBMC cultures using peptide 154-163.

Some of these T cell lines have been phenotyped. As seen in Table 8, onecell line designated T-866 (T-Donor A), which was derived from pulsingwith peptide 141-150, contains appreciable amounts of CD4+/CD8+ doublepositive cells and another cell line, designated T-1538 (T Donor B),derived from pulsing with peptide 154-163, shows a similar phenotype.

Four of the T cell lines derived from three different individuals werethen assays for their ability to lyse human cells (Table 9). As seen inTable 9, the T cell line designated T-866, derived from peptide 141-150,was able to lyse T2 cells when pulsed with the appropriate peptide(141-150). No lysis was seen using the PSA negative human colon cancercell line COLO-205. While 80% lysis was seen using the LNCAP PSAexpressing human prostate cancer cell line. When employing the NK targetK562, which measures non-specific lysis due to NK cell activity, only23% lysis was obtained. Similar results were seen employing a differentT cell line obtained from the same patient which was derived frompulsing with PSA peptide 154-163. Two additional T cell lines which werederived from peptide 154-163 were also analyzed. One was from a normaldonor (T-1538) and one was from a prostate cancer patient (T-PC2; TDonor C). As can be seen in Table 9, employing both of these T celllines, enhanced lysis was seen when the T2 cell line was pulsed with the154-163 peptide and enhanced lysis was seen when employing the PSAexpressing prostate specific cell line LNCAP, as compared to COLO-205 orK562. These studies demonstrate that T cell lines can be establishedusing the peptides and protocols generated here which have the abilityto lyse PSA expressing human prostate carcinoma cells.

TABLE 8 Flow cytometry analysis of PSA peptide specific T cells PSAT-cell Line Peptide CD3 CD4 CD8 CD4/CD8 CD56 T-Donor A 141-150 96 35 6.559 0 T-Donor B 154-163 94 5.2 32 62 0 Results are expressed in %positive cells.

TABLE 9 Cytotoxic effects of PSA peptide specific T cells % specificrelease (lysis) T-cell PSA T2 + Line Peptide T2 peptide LNCAP K562COLO-205 T-Donor A 141-150  10^(a) 40^(b) 80^(b) 23 7 T-Donor A 154-16016 35^(b) 60^(b) 22 10 T-Donor B 154-160 10 40^(b) 29^(b) 3 10 T-Donor C154-160 15 35^(b) 35^(b) 2 8 ^(a)Percent of ¹¹¹ In specific release 24hour cytotoxic assay (E:T ratio, 25:1) ^(b)p < 0.01 significant

Example IV Construction and Characterization of Prostate SpecificAntigen Oligo-Epitope Peptide

Two 10-mer PSA peptides (PSA1 and PSA3) which were selected to conformto human HLA class 1-A2 motifs elicited PSA specific CTL responses inboth normal donors and patients with prostate cancer. A longer PSApeptide (30-mer), designated prostate specific antigen oligo-epitopepeptide (PSA-OP) comprising the shorter PSA-1 and PSA-3 peptidesequences, was investigated for the ability to mediate PSA specificcytotoxic T-cell activity (Table 10).

TABLE 10 Sequence of 30 amino acid PSA peptide PSA-OP FLTPKKLQCV/   DLH/VISNDVCAQV  /HPQKVTK (Seq. ID No: 4) (141-170) PSA-1 FLTPKKLQCV (Seq. IDNo: 1) (141-150) PSA-3               VISNDVCAQV (Seq. ID No: 2)(154-163)

Materials and Methods Peptide Synthesis

PSA-OP was synthesized on an Applied Biosystems Model 432A peptidesynthesizer. It operates on a 25 μmole scale and employs f-moc chemistryand feedback monitoring to control the coupling of each successive aminoacid to the growing chain. The completed peptide is cleaved off thesynthesis resin with trifluoroacetic acid and thioanisole/ethanedithiolas scavengers. Acid salts are extracted with tert-butyl methyl ether andthe peptide is lyophilized from water to give approximately 64 mgpowder. The powder is soluble at 2 mg/ml in 1% DMSO and sterile-filteredaliquots show a single sharp peak on C18 reverse phase high performanceliquid chromatography.

Cell Cultures

Prostate carcinoma cell lines LNCAP and DU-145 [HLA-A2+] were purchasedfrom American Type Culture Collection (Rockville, Md.). Cultures weremycoplasma free and were maintained in complete medium, Dulbecco'smodified Eagle medium and RPMI1640 medium, respectively (LifeTechnologies Inc. GIBCO BRL, Grand Island, N.Y.) supplemented with 10%fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100μ/ml streptomycin (Life Technologies, Inc.). The 174CEM.T2 cell line(T2) (transport deletion mutant) is described in Anderson et al, 1993.CIR-A2 cell line is described in Storkus et al, 1987. T2 cell and CIRA2cells were maintained in Iscove's modified Dulbecco's complete medium(IMDM) and RPMI1640 complete medium, respectively.

Generation of T Cell Lines

Peripheral blood mononuclear cells (PBMCs) were obtained fromheparinized blood of healthy HLA-A2 donors using a lymphocyte separationmedium gradient (Organon Technika, Durham, N.C.). The mononuclear cellfraction was washed 3 times and PBMCs were resuspended in completemedium: AIM V (Life Technologies, Inc.) supplemented with 5% human ABserum (Valley Biomedical, Winchester, Va.), 2 mM glutamine, 100 U/ml ofpenicillin, and 100 μg/ml of streptomycin (GIBCO). Cells (2×10⁵) incomplete medium in a volume of 100 μl were put into each well of a96-well flat-bottom assay plate (Corning, Costar Corp., Cambridge,Mass.). Peptides were added to cultures at a final concentration of 50μl/ml. Cultures were incubated for 5 days at 37° C. in a humidifiedatmosphere containing 5% CO₂. After removal of the peptide-containingmedium, the cultures were then provided with human IL-2-(Cetus) (20U/ml) for 11 days, with IL-2-containing medium being replenished every 3days. The incubation time of 5 days with peptide plus 11 days with IL-2constitutes one cycle. Primary cultures were restimulated with the samepeptide (50 μg/ml) on day 1 of each cycle. Irradiated (4,000 rads)autologous PBMCs (1×10⁶) were added in a volume of 100 μl in completemedium (AIM-V) and used as antigen presenting cells.

Cytotoxic Assays

Various target cells were labeled with 50 μCi of ¹¹¹In-oxyquinoline(Medi-Physics Inc., Arlington, Ill.) for 15 minutes at room temperature.Target cells (0.2×10⁴) in 100 μl of complete medium (see below) wereadded to each of 96 wells in flat bottom assay plates (Corning CostarCorp.). The labeled targets were incubated with peptides at a finalconcentration of 50 μg/ml for 60 min at 37° C. in 5% CO₂ before addingeffector cells. Effector cells were suspended in 100 μl of completemedium supplemented with 5% pooled human AB serum and added to targetcells. The plates were incubated at 37° C. for 16 hours. Supernatantswere harvested for γ-counting using harvester frames (Skatron, Inc.Sterling, Va.). Determinations were carried out in triplicate andstandard deviations were calculated. All experiments were carried outthree times. Specific Lysis was calculated using the following formula:

${\% \mspace{14mu} {of}\mspace{14mu} {Specific}\mspace{14mu} {Release}} = \frac{{{observed}\mspace{14mu} {{release}({cpm})}} - {{spontaneous}{\mspace{11mu} \;}{release}\mspace{14mu} ({cpm})}}{{{total}{\mspace{11mu} \;}{releae}\mspace{14mu} ({cpm})} - {{spontaneous}\mspace{14mu} {release}\mspace{14mu} ({cpm}) \times 100}}$

Spontaneous release was determined from wells to which 100 μl ofcomplete medium, instead of effector cells was added. Total releasableradioactivity was obtained after treatment of target cells with 2.5%Triton X-100.

Experiments using protease inhibitors were performed using C1R-A2 cellsas target cells pulsed with 100 μg/ml of PSA-OP for 3 hr. Peptide pulsedtarget cells were incubated with protease inhibitors. Proteaseinhibitors used were E64, carboxypeptidase inhibitor, plummer inhibitor,and captopril (angiotension converting enzyme inhibitor) at variousconcentrations (10⁻⁵, 10⁻⁶, 10⁻⁷ M). CTL activity was determined by theCTL assay described above.

Limiting Dilution Analysis

Limiting dilution assays were used for the determination of precursorfrequencies to PSA-1, PSA-3 and PSA-OP. Various number of PBMCs wereseeded in 96-well flat bottom plates (Corning Costar) with 1×10⁴autologous PBMC irradiated with 4,000 rads and incubated with 50 μg/mlof PSA peptide. Cells were cultured in complete medium as describedpreviously. At least 48 cultures were set up for each dilution. Cultureswere incubated for 5 days at 37° C. in a humidified atmospherecontaining 5% CO₂ and then provided with fresh medium containing humanIL-2 for 11 days, with IL-2-containing medium being replenished every 72hr as shown below for CTLs generation. After two cycles of stimulationspecific cytotoxic activity was tested for each single well, againstCIR-A2 target cells with or without incubation with peptide. Cytotoxicassays were similar to the method described above. The unlabelled K562cells were added to the microwells containing responder cells. After 1hr incubation at 37° C. target cells were added into each well andincubated for 6 hrs at 37° C. Precursor frequencies were calculated byX² minimization.

Flow Cytometry

Single-color flow cytometric analysis: 1×10⁶ cells were washed threetimes with cold Ca²⁺- and Mg²-free Dulbecco's phosphate-buffered saline(DPBS) and then stained for 1 hr with 1 μg of monoclonal antibody (MAb)against CD3, CD4, CD8, CD56, CD19, HLA class II (HLA-DR) (BectonDickinson, San Jose, Calif.). HLA class I (W6/32) (Seratec, Sussex,England), and MOPC-21 (Cappel/Organon Teknika Corp., West Chester, Pa.)in a volume of 100 μl of PBS containing 1% bovine serum albumin. Thecells were then washed three times with cold DPBS and incubated for anadditional hour in the presence of 1:100 dilution (volume of 100 μl PBScontaining 1% bovine serum albumin) of fluorescein-conjugated goatanti-mouse immunoglobulin (Ig) (Kirkeggard and Perry Labs,Gaitheresburg, Md.). The cells were again washed three times with DPBSand resuspended in DPBS at the concentration of 1×10⁶ cells/ml. Thecells were immediately analyzed using a Becton Dickinson FACScanequipped with a blue laser with an excitation of 15 nW at 488 nm. Datawere gathered from 10,000 live cells and used to generate results.

Dual color flow cytometric analysis: The procedure for two-color flowcytometry analysis was similar to that used for single-color analysiswith the following exceptions. The MAbs used were anti-CD4 fluoresceinconjugate, anti-CD8 phycoerythrin conjugate, anti IgG1 fluoresceinconjugate and anti-IgG2a phycoerythrin conjugate (Becton Dickinson).Staining was done simultaneously for 1 hr after which cells were washedthree times, resuspended as above, and immediately analyzed using aBecton Dickinson FACSort equipped with a blue laser with an excitationof 15 nW at 488 nm and the Lysis II program.

Peptide Binding to HLA-A2

Binding of PSA-1 and PSA-3 and PSA-OP peptides to the HLA-A2 moleculeswas evaluated by upregulation of the expression of these molecules onthe cell surface of T2 cells as demonstrated by flow cytometry. 1×10⁶cells in serum-free IMDM were incubated with peptides at theconcentration of 50 μg/ml in 24-well culture plates at 37° C. in 5% CO₂.Flow cytometry for peptide binding was performed using T2 cells andsingle color analysis. After cells were washed three times in DPBS asabove, they were incubated for 1 hr with HLA-A2 specific MAb A2,69 (Onelambda, Inc., Canoga Park, Calif.), using 10 μl of a 1× working dilutionper 10⁶ cells. MOPC-21 (Cappel/Organon Teknika Corp.) was used asisotype control. The cells were then washed three times and incubatedwith 1:100 dilution of fluorescein (FITC) labeled anti-mouse IgG(Kirkegaard & Perry, Gaithersburg, Md.). Analysis was carried out usingthe FACScan as described above. Cells were maintained on ice during allcell preparation and staining unless otherwise stated.

HLA Typing

The HLA phenotyping of donor A (HLA-A2,24; B27,35; C2,4; DR B1*0101,B1*1104; DQw B10501, B1*0301; DRwB3*0202) and donor B (HLA-A2, 29;B7,44; Cw5-; DR13,-; DQw6,-; DRw52,-) was performed by the Blood Bank ofNIH on PBMC using a standard antibody-dependent micro-cytotoxicity assayand a defined panel of anti-HLA antisera or DNA assay. The following HLAphenotypes were found for healthy donors utilized for PSA (rV-PSA) wasgenerated by the methods described in Hodge et al, 1995. The PSA genewas isolated as a complementary DNA (cDNA) clone from a human prostatecarcinoma cell cDNA library. The PSA cDNA was inserted under the controlof the vaccinia 40K promoter into the Hind III M region of the genome ofthe attenuated Wyeth strain vaccine virus.

A recombinant vaccinia virus expressing PSA-OP was made by the samemethodology (Hodge et al, 1995).

Vaccinia Virus Infection of Prostate Carcinoma Cells

DU-145 target cells at the concentration of 1×10⁷ per ml in completeRPMI-1640 medium supplemented with 0.1% bovine serum albumin wereincubated with an equal volume of vaccinia virus (10 MOI) in the samemedium at 37° C. for 1.5 hours. The cells were then seeded at 10⁵ cellper ml in complete medium with 10% FBS, into a 24-well culture plates at37° C. in 5% CO₂ for 24 hr before being utilized as targets in cytotoxicassay experiments, bPSA production from rV-PSA vaccinia virus wasevaluated by immunoradioassay kit purchased from Tandem Co.

Statistical Analysis

Statistical analysis of differences between means was done by a twotailed paired t-test.

Results

Two T-cell lines from different normal donors were established by invitro stimulation with PSA-OP. The T-cell lines were phenotypicallyCD4⁺, CD8⁺, or CD4⁺/CD8⁺ and CD56⁻ as shown in Table 11.

TABLE 11 Flow Cytometric Analysis Of Surface Markers On T Cell Lines TCell Line CD3+ CD4+/CD8− CD4−/CD8+ CD4+/CD8+ CD56+ T/PSA-OP-193.80(94.10)  5.83(109.00) 32.00(4550.00) 62.00(139/110) neg T/PSA-OP-295.86(167.77) 52.38(2505.00) 41.55(2151.00)  4.46(153/3325) neg Resultsare expressed as percentage of fluorescent cells and (x/y) meanfluorescent intensity per cell. Marker expression was considerednegative (neg)when lower that 4%. Results are expressed as a percentageof each T-cell line reactive with mAbs. Routinely, 2.0-4.0% of cells arestained when treated either with no priming MAbs or an isotype relatedcontrol MAb.

The human CTLs lysed PSA-OP as well as PSA-1 or PSA-3 pulsed CIR-A2cells as shown in Table 12.

TABLE 12 CTL activity of PSA-OP specific T cell lines Donor No peptidePSA-OP PSA-1 PSA-2 PSA-3 T/PSA-OP-1 7.9(2.7) 32.3*(0.87) 8.4(4.0)1.8(0.30) 28.7*(1.12) T/PSA-OP-2  0.0(0.06) 16.5*(1.0)  14.3*(0.6) 6.4(1.40) 16.5*(1.0)  T/PSA-OP-2 specifically lysed CIR-A2 cells pulsedwith PSA-OP, PSA-1 and PSA-3 peptides, while T/PSA-OP-1 cell line onlylysed CIRA2 when pulsed with PSA-OP and PSA-3. CIRA2 cells have beenpulsed with 50 μg/ml of PSA peptide for 3 h, before to be labeled with¹¹¹ In and utilized as target in 18 h CTL cytotoxic assay. Results areexpressed as % of Specific release (SD) at the E:T ratio of 25:1. *P <0.02

The human CTLs also lysed PSA⁺ HLA-A2⁺ human prostate cancer cells asshown in Table 13.

TABLE 13 CTL activity of PSA-OP specific T cell lines against HLA-A2001+, PSA-producing human Prostate Carcinoma Cells LNCAP Donor 12.5:125:1 E:T ratio T/PSA-OP-1 32.54*(6.6) 50.4*(6.4) T/PSA-OP-2 14.60*(3.7)24.8*(1.6) Established CTLs from Donors 1 & 2 lyse LNCAP Prostatecarcinoma cells. 10⁶ LNCAP cells have been labelled with ¹¹¹In and usedas targets in 18 h CTL cytotoxic assay. Results are here expressed as %of Specific Release (SD). P < 0.016.

The HLA-A2⁺ DU-145 prostatic carcinoma cell line was infected withvaccinia virus engineered with the PSA gene or the PSA-OP gene. TheDU-145 cells infected with the rV-PSA vaccinia virus expressed PSA asshown in Table 14.

TABLE 14 PSA production by DU-145 infected with wt and rV-PSA vacciniavirus 4 hours 24 hours Prostatic Carcinoma Cells [pg/ml] [pg/ml] DU-145(WT) 1.2 (0.15)  1.7 (1.20) DU-145 (rV-PSA) 5.1 (0.50) 12.0 (4.50) LNCAPND 232.0 (4.60)  DU-145 cells production of PSA after rV-PSA vacciniavirus infection. DU-145 have been incubated with wild type of rV-PSAVaccinia virus (10 MOI) for 4 and 24 h before the supernatant washarvested and evaluated for PSA production by IRMA detection PSA kit(tandem). LNCAP prostate cancer carcinoma cells have been used as apositive control since they are known to produce large amounts of PSA in24 h. Results are expressed as pg/ml (SD) per 10⁶ of cells.

The ability of the human PSA-OP specific T lymphocyte cell lines to lysethe rV-PSA or rV-PSA-OP vaccinia virus infected DU-145 prostatic cellswas determined. As Table 15 shows, the human T-cell lines lysed rV-PSAinfected targets as well as rV-PSA-OP infected targets.

TABLE 15 CTL activity of PSA-OP specific T cell lines againstHLA-A2001 + DU-145 prostate cell line, infected with vaccinia virus,engineered with PSA gene and PSA-OP mini-gene DU-145 DU-145 DU-145 [WT][rV-PSA] [rV-PSA-OP] T Cell Line 12.5:1 25.1 12.5:1 25:1 12.5:1 25:1 E:Tratio T/PSA-OP-1 15.0(3.8) 26.5(6.1) 31.8*(5.0) 45.7*(6.1) 32.5*(6.6)50.4*(6.4) T/PSA-OP-2  0.0(2.0)  6.2(4.9)  4.0*(2.1) 10.5*(2.8)10.9*(1.4) 14.6*(3.7) Established CTLs lysed DU-145 prostate cancercells after infection with rV-PSA and rV-PSA-OP vaccinia virus. DU-145cells have been incubated with Vaccinia virus (10 MOI) wild type orcarrying the PSA gene or PSA-OP mini-gene for 24 h before being labeledand incubated with effector cells. The ability of rV-PSA infected cellsto produce PSA was evaluated by a radio immunoassay kit from Tandem.Results are here expressed as % of Specific Release (SD). *P < 0.05.

The ability of PSA-OP to directly bind to a HLA Class I-A2 molecule wasdetermined. The results in Table 16 showed that PSA-OP did not bindHLA-A2 as indicated by the lack of upregulation of A2 expression on 174CEM-12 cells.

TABLE 16 Binding of PSA peptides to the HLA class-I A2001 moleculePeptide #T2 binding *Predicted binding None 16.88 Neg PSA [42-51] 51.44Neg PSA-OP 63.28 Neg PSA-1 127.73 Pos PSA-3 123.71 Pos MTX [58-66]157.86 Pos PSA-OP does not bind the HLA class-I A2001 molecules on T2cell surface *Predicted binding on the basis of published motifs: Pos =positive Neg = negative #Reaction of T2 cells with anti-HLA-A2 mAb afterthe cells had been incubated for 24 h with control peptides and PSApeptides (50 μg/ml/10⁶ cells), PSA [42-51] was considered as a negativecontrol peptide, since it is unable to bind HLA class I-A2, while PSA-1,PSA-3, MTX [58-66] were considered as a positive control. The resultsare expressed as relative fluorescence (mean intensity) and 100 wasarbitrarily chosen as a cut off value for positivity.

Since it was shown that the 30-mer PSA-OP peptide did not directly bindto HLA class I-A2 molecules but did stimulate cytotoxic cells to lysePSA⁺ HLA class I-A2 targets, studies were undertaken to determine if the30-mer PSA-OP peptide was cleaved to smaller peptides by proteolyticactivity to allow it to interact with HLA class I-A2 molecules.

The effects of protease inhibitors on CTL mediated killing of CIR-A2cells pulsed with PSA-OP peptide was determined. The results shown inTable 17 show decreased cytotoxicity in the presence of proteaseinhibitors. These results indicate that the PSA-OP is cleaved byproteases on the cell surface of the target cells into shorter peptideswhich, in turn, interact with HLA-A2 molecules and as a consequenceinduce specific CTL lysis of CIR-A2 target cells.

TABLE 17 Effects of protease inhibitors on CTL mediated killing of CIRA2Cells Pulsed with PSA-OP Peptide Donor-1 PSA-OP PSA-OP peptide PeptidesPulsed in Human Serum No peptide [50 μg/ml] Medium  7.7 (1.9) 26.1*(4.3)  E 64 [10−⁶M] 11.9 (1.7) 13.7 (0.6) Plummer's [10⁻⁵M] 10.7 (2.0)18.7* (2.4)  Captopril [10⁻⁶M] 15.8 (2.1) 11.0 (2.6) PCI [10⁻⁵M] 13.2(1.6) 13.7 (3.2) Extracellular carboxypeptidase inhibitors (E64,captopril and PCI) block the lysis of CIR-A2 cells pulsed with PSA-OPpeptide by established CTLs. Results are expressed as % of SpecificRelease (+/−SD) at 25:1 E:T ratio. *P < 0.03.

Precursor frequency (PF) studies showed that PF for PSA-1, alone, andPSA-3, alone, varied from donor to donor. In contrast, the PF for PSA-OPwas strikingly similar from donor to donor as shown in Table 18.

TABLE 18 Precursor Frequency Study % of negative microcolonies Number ofDonor 10,000 5,000 1,000 500 precursors T/PSA-OP 2 donor PSA-OP 91.689.3 100.0 100.0 1/80006 PSA-1 47.9 75.0 93.7 95.8 1/14695 PSA-3 95.897.8 100.0 100.0  1/244200 T/PSA-OP 3 donor PSA-OP 93.7 95.8 95.8 96.91/70229 PSA-1 93.7 97.9 100.0 100.0  1/181480 PSA-3 89.6 93.7 95.8 97.61/59279 CTL precursors specific for PSA-OP, PSA-1 and PSA-3 peptides arepresent in PBLs isolated from male HLA-A2001⁺ donors after two cycles ofstimulation with PSA peptides. Precursors' cytotoxic effects wereevaluated against CIRA2 pulsed for 3 h with respective PSA peptides inthe presence of cold K562 (10,000/well).

Thus, PSA-OP as an immunogen offers distinct advantages over the use ofPSA-1 alone or PSA-3 alone in eliciting consistent numbers of cytotoxicT lymphocytes from individual to individual. Furthermore, PSA-OPcomprises more than one epitope peptide sequence which may fit theconsensus motif for a variety of HLA-class I molecule types includingHLA-A2, A3, A11, Aw68 and B53. HLA-A2 is present in approximately 50% ofNorth American Caucasians and 34% of African Americans. HLA-A3 ispresent in 26 and 17% of North American Caucasians and AfricanAmericans, respectively. HLA-A11 is present in 40% of the Asianpopulation and HLA-B53 is present in 22% of African Americans.Consequently, PSA-OP may be useful as an immunogen in eliciting PSAspecific immune responses in a broad segment of the human population.

This invention has been described in detail including the preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements thereon without departing from the spiritand scope of the invention as set forth in the claims.

References and patents referred to are incorporated herein by reference.

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1.-41. (canceled)
 42. A method for eliciting an immune response againstprostate specific antigen for treating a human comprising: (a)administering to the human a priming inoculation of an effective amountof a first recombinant virus, wherein the virus is characterized byencoding a prostate specific antigen oligo-epitope peptide (PSA-OP); and(b) administering to the human one or more boosting inoculations of aneffective amount of a second recombinant virus of a different genus,wherein the virus is characterized by encoding a prostate specificantigen oligo-epitope peptide (PSA-OP).
 43. The method of claim 42,wherein the first recombinant virus is an orthopox virus encoding aprostate specific antigen oligo-epitope peptide (PSA-OP) and the secondrecombinant virus is an avipox virus encoding a prostate specificantigen oligo-epitope peptide (PSA-OP).
 44. The method of claim 42,wherein the first recombinant virus is vaccinia encoding a prostatespecific antigen oligo-epitope peptide (PSA-OP) and the secondrecombinant virus is fowlpox encoding a prostate specific antigencomprising an oligo-epitope peptide (PSA-OP).
 45. The method of claim42, wherein the PSA-OP consists of the amino acid sequence: (SEQ ID NO:4) (a) F   L   T   P   K   K   L   Q   C   V   D   L 141 142 143 144 145146 147 148 149 150 151 152H   V   I   S   N   D   V   C   A   Q   V   H 153 154 155 156 157 158159 160 161 162 163 164 P   Q   K   V   T   K or

(b) SEQ ID NO:4 wherein valine is substituted for an amino acid residueat one or more positions selected from the group consisting of position148, position 149, position 160, and position 161, or wherein at leastone of the amino acids at positions 151, 152, and 153 is deleted. 46.The method of claim 42, wherein the recombinant virus further comprisesa DNA sequence encoding an immunoenhancing molecule selected from thegroup consisting of influenza peptide, tetanus toxoid, tetanus toxoidCD4-epitope, Pseudomonas exotoxin A, and poly-L-lysine.
 47. The methodof claim 42, wherein the recombinant virus is administered at a dose ofabout 0.5 mg to about 100 mg per kilogram of the human.
 48. The methodof claim 42, wherein the recombinant virus is administered at a dose of10⁵ pfu-10⁹ pfu.
 49. The method of claim 42, wherein the recombinantvirus is administered at a dose of 10⁷ pfu.
 50. The method of claim 42,wherein the second recombinant virus is administered at periodicintervals after the first recombinant virus.
 51. The method of claim 49,wherein the second recombinant virus is administered as several dosesafter the first recombinant virus at a periodic interval of 1-3 months.52. A method for stimulating the immune system of a human againstprostate specific antigen to prevent the establishment and growth of PSApositive carcinoma cells comprising: (a) administering to the human apriming inoculation of an effective amount of a first recombinant virus,wherein the virus is characterized by encoding a prostate specificantigen oligo-epitope peptide (PSA-OP); and (b) administering to thehuman one or more boosting inoculations of an effective amount of asecond recombinant virus of a different genus, wherein the virus ischaracterized by encoding a prostate specific antigen oligo-epitopepeptide (PSA-OP), wherein the recombinant virus causes the expression ofthe PSA-OP in the human in the amount sufficient to effect thestimulation.
 53. The method of claim 52, wherein the first recombinantvirus is an orthopox virus encoding a prostate specific antigenoligo-epitope peptide (PSA-OP) and the second recombinant virus is anavipox virus encoding a prostate specific antigen oligo-epitope peptide(PSA-OP).
 54. The method of claim 52, wherein the first recombinantvirus is vaccinia encoding a prostate specific antigen oligo-epitopepeptide (PSA-OP) and the second recombinant virus is fowlpox encoding aprostate specific antigen oligo-epitope peptide (PSA-OP).
 55. The methodof claim 52, wherein the PSA-OP consists of the amino acid sequence:(SEQ ID NO: 4) (a) F   L   T   P   K   K   L   Q   C   V   D   L 141 142143 144 145 146 147 148 149 150 151 152H   V   I   S   N   D   V   C   A   Q   V   H 153 154 155 156 157 158159 160 161 162 163 164 P   Q   K   V   T   K or

(b) SEQ ID NO:4 wherein valine is substituted for an amino acid residueat one or more positions selected from the group consisting of position148, position 149, position 160, and position 161, or wherein at leastone of the amino acids at positions 151, 152, and 153 is deleted. 56.The method of claim 52, wherein the recombinant virus further comprisesa DNA sequence encoding an immunoenhancing molecule selected from thegroup consisting of influenza peptide, tetanus toxoid, tetanus toxoidCD4-epitope, Pseudomonas exotoxin A, and poly-L-lysine.
 57. The methodof claim 52, wherein the recombinant virus is administered at a dose ofabout 0.5 mg to about 100 mg per kilogram of the human.
 58. The methodof claim 52, wherein the recombinant virus is administered at a dose of10⁵ pfu-10⁹ pfu.
 59. The method of claim 52, wherein the recombinantvirus is administered at a dose of 10⁷ pfu.
 60. The method of claim 52,wherein the second recombinant virus is administered at periodicintervals after the first recombinant virus.
 61. The method of claim 60,wherein the second recombinant virus is administered as several dosesafter the first recombinant virus at a periodic interval of 1-3 months.62. A kit for treating a human comprising: (a) an effective amount of afirst recombinant virus, wherein the virus is characterized by encodinga prostate specific antigen oligo-epitope peptide (PSA-OP), (b) aneffective amount of a second recombinant virus of a different genus,wherein the virus is characterized by encoding a prostate specificantigen oligo-epitope peptide (PSA-OP), and (c) instructions toadminister the first recombinant virus in a priming inoculation and thesecond recombinant virus in one or more boosting inoculations.
 63. Thekit of claim 62, wherein the first recombinant virus is an orthopoxvirus encoding a prostate specific antigen oligo-epitope peptide(PSA-OP) and the second recombinant virus is an avipox virus encoding aprostate specific antigen oligo-epitope peptide (PSA-OP).
 64. The kit ofclaim 62, wherein the first recombinant virus is vaccinia encoding aprostate specific antigen oligo-epitope peptide (PSA-OP) and the secondrecombinant virus is fowlpox encoding a prostate specific antigenoligo-epitope peptide (PSA-OP).
 65. The kit of claim 62, wherein thePSA-OP consists of the amino acid sequence: (SEQ ID NO: 4) (a)F   L   T   P   K   K   L   Q   C   V   D   L 141 142 143 144 145 146147 148 149 150 151 152 H   V   I   S   N   D   V   C   A   Q   V   H153 154 155 156 157 158 159 160 161 162 163 164 P   Q   K   V   T   K or

(b) SEQ ID NO:4 wherein valine is substituted for an amino acid residueat one or more positions selected from the group consisting of position148, position 149, position 160, and position 161, or wherein at leastone of the amino acids at positions 151, 152, and 153 is deleted. 66.The kit of claim 62, wherein the recombinant virus further comprises aDNA sequence encoding an immunoenhancing molecule selected from thegroup consisting of influenza peptide, tetanus toxoid, tetanus toxoidCD4-epitope, Pseudomonas exotoxin A, and poly-L-lysine.
 67. The kit ofclaim 62, wherein the instructions indicate the recombinant virus isadministered at a dose of about 0.5 mg to about 100 mg per kilogram ofthe human.
 68. The kit of claim 62, wherein the instructions indicatethe recombinant virus is administered at a dose of 10⁵ pfu-10⁹ pfu. 69.The kit of claim 62, wherein the instructions indicate the recombinantvirus is administered at a dose of 10⁷ pfu.
 70. The kit of claim 62,wherein the instructions indicate the second recombinant virus isadministered at periodic intervals after the first recombinant virus.71. The kit of claim 70, wherein the instructions indicate the secondrecombinant virus is administered as several doses after the firstrecombinant virus at a periodic interval of 1-3 months.